Laser generated micro- and nanoeffects: Inactivation of proteins coupled to gold nanoparticles with nano- and picosecond pulses

Background: Protein denaturation in the fs-ns time regime is of fundamental interest for high precision applications in laser tissue interaction. Conjugates of colloidal gold coupled to proteins are presented as a model system for investigating ultrafast protein denaturation. It is expected that irradiation of such conjugates in tissue using pi coup to nanosecond laser pulses could result in effects with a spatial confinement in the regime of single macromolecules up to organelles. Materials and Methods: Experiments were done with bovine intestinal alkaline phosphatase (aP) coupled to 15 nm colloidal Gold. This complex was irradiated at 527 nm/ 532 nm with a variable number of pico- and nanosecond pulses. The radiant exposure per pulse was varied from 2 to 50 mJ/cm² in the case of the picosecond pulses and 10 to 500 mJ/cm² in the case of the nanosecond pulses. Denaturation was detected as a loss of protein function with the help of the fluorescence substrate 4MUP. Results and Discussion: Irradiation did result in a steady decrease of the aP activity with increasing radiant exposures and increasing number of pulses. Inactivations up to 80% using 35ps pulses at 527 nm with 50mJ/cm² and a complete inactivation induced by 16 ns pulses at 450 mJ/cm² are discussed. The induced temperature 111 the particles and the surrounding water was calculated using Mie’s formulas for the absorption of the nanometer gold particles and an analytical solution of the equations for heat diffusion. The calculated temperatures suggest that picosecond pulses heat a molecular scaled area whereas nanosecond pulses could be used for targeting larger cellular compartiments. It is difficult to identify one of the possible damage mechanisms, i.e. thermal denaturation or formation of micro bubbles, from the dependance of the inactivation 011 pulse energy and number of applied pulses. Therefore experiments are needed to further elucidate the damage mechanisms. The observed inactivation dependencies on applied energy and radiant power can not be explained with one or two photon photochemistry. In conclusion, denaturing proteins irreversibly via nanoabsorbers using pico-/ nanosecond laser pulses is possible. The expected confinement of the heat to the nanoabsorbers suggests that denaturation of proteins with nanometer precision could be possible with this approach. However, the mechanism of protein inactivation, which is part of present investigations, is crucial for the precision of such nanoeffects.